A New Technique to Spatiotemporally Resolve Protein Interaction Networks in Living Cells

Roughly 800 different types of GPCR play crucial roles throughout the body, including regulating heart rate, blood pressure and digestion; mediating the senses of sight, smell, and taste; and enabling many forms of chemical communication between cells in the brain. Approximately 40 percent of medicines target one type of GPCR or another, including schizophrenia drugs that target dopamine receptors, painkillers that target opioid receptors, and allergy and heartburn drugs that target different types of histamine receptors, just to name a few.

These many types of GPCR have one feature in common that makes them particularly difficult to study: when they are activated (whether by a beam of light or a blood-borne hormone), they set off a rapid cascade of biochemical reactions, in which the GPCRs themselves physically move from one location to another within the cell and trigger signals that are passed among dozens or hundreds of different protein messengers. Together, these changes end up altering a cell's behavior -- for example changing the excitability of neurons or reprogramming their genetic activity.

The new technology, described in a paper published in Cell, makes it vastly easier for scientists to study the GPCRs. In a first proof-of-principle study, the team used their new approach to identify new biochemical players involved in the development of tolerance to opioid painkillers -- which target a particular type of GPCR -- findings they anticipate will enable researchers to develop safer and more effective pain control.

"The methodology that our collaborative team developed allows one to precisely define the local protein environment of receptors as they dynamically change partners and move within the cell," said paper's one of the senior authors. "We ourselves were surprised by the high degree of spatial and temporal resolution that this methodology can achieve."

Starting with a list of proteins that are known collaborators of a particular GPCR, researchers trigger GPCR activity and use a biochemical tracking device to identify these proteins' associates in other parts of the cell.

To build this network of associates, the researchers turned their receptor of interest into an "informant" by outfitting it with a tracking device in the form of an enzyme called APEX, which can be triggered to spray any nearby proteins with a chemical tag. Researchers can then use this tag to track down and identify suspected participants in the GPCR cascade using a technique called mass spectrometry. By triggering APEX tagging at different times after activating the GPCR, the researchers were able to begin building a detailed and unbiased map of the protein network underlying a cell's response to activation of a particular GPCR.

In a proof-of-principle experiment, the team used their technique to answer a long-standing mystery about the biological mechanisms of opioid tolerance -- the phenomenon by which, over time, patients tend to need higher and higher doses of opioid painkillers such as morphine to achieve the same level of pain management.

This is an important puzzle to solve, because increased opioid use in response to tolerance puts patients at risk of serious adverse side effects and also promotes addiction. Researchers know that tolerance occurs when cells respond to long-term opioid use by destroying or "down-regulating" the GPCR opioid receptors that these drugs target, but what triggers cells to do this is unknown.

Using their APEX-based tool, the researchers found that two cellular proteins not previously known to interact with opioid receptors in fact partner closely with delta-opioid receptors (a subtype of opioid receptor) at precisely the time and place at which the cell targets these receptors for destruction. They then confirmed, using genetic manipulations, that both proteins are essential for the down-regulation process.

Researchers emphasize that not all suspects revealed by their technique will prove to be important in a given GPCR cascade. But the ability to quickly and easily identify likely participants in a given cascade should dramatically quicken the pace toward understanding these complex signaling processes, and to develop more targeted treatments for diseases in which they go awry.